2-Methylthiazolidine-2,4-Dicarboxylic acid and salts thereof for the treatment of neurodegenerative diseases

ABSTRACT

The invention relates to the compounds (2R,4R), (2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and (2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and salts of said compounds, the use thereof for the prophylaxis and/or treatment of neurodegenerative diseases, methods for the prophylaxis and/or treatment of neurodegenerative diseases, and pharmaceutical compositions containing said compounds together with physiologically compatible carriers, auxiliary agents, and/or solvents.

The invention relates to the compounds (2R,4R)-,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid as well as(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and salts of thesecompounds, their use for prophylaxis and/or treatment ofneurodegenerative diseases, methods for prophylaxis and/or treatment ofneurodegenerative diseases and pharmaceutic formulations, which comprisesaid compounds and/or salts thereof optionally together with furtheradditives.

Cerebral circulatory disorders, ischemia, cerebral ischemia, apoplexy,cerebral apoplexy, brain deficiencies, senile dementia, Alzheimer'sdisease, Huntington's chorea and Parkinson's disease for example arecounted among the neurodegenerative diseases.

The Parkinson's disease belongs to the frequent neurodegenerativediseases of senior persons.

The Parkinson's disease (or idiopathic Parkinson's syndrome) is aprogressive disease, which is characterized by the symptoms of restingtremor, hypo-/bradykinesia and rigidity of the musculature.Neuropathologically viewed, a degeneration of the dopaminergic neurons,which project to the striatum, occurs in the substantia nigra withdecrease of the striatal dopamine concentration as well as hyalineinclusion bodies (Lewy bodies) in the remaining neurons. Etiology andpathophysiology of this disease are widely unknown. The idiopathicParkinson's syndrome accounts to the frequent neurological diseases ofsenior persons. The first symptoms occur usually after the age of 50years, young persons are affected very seldom. The prevalence increasesexponentially after the age of 60 years, such that about 1 to 1.5% ofthe persons above the age of 60 years are affected.

Via a symptomatic treatment by substitution of the dopamine deficiency,a treatment of the symptoms is possible for a period of 5 to 10 years.But at present the progression of the disease, i.e. the degenerationvelocity of the dopaminergic neurons, cannot be influenced. Theinitially positive reports about a neuroprotective effect of theirreversible monoamine oxidase B-inhibitor selegiline did not approve inthe long-term devolution.

Today, 3 categories of pharmaceuticals are available for themedicamentous Parkinson therapy.

Dopaminergics

-   L-DOPA plus decarboxylase inhibitor (e.g. Madopar®, Nacom®)-   Dopamine agonists: bromocriptine (Pravidel®), lisuride (Dopergin®),    pergolide (Parkotil®), dihydrocryptine (Almirid®), cabergoline,    pramipexole, ropinirole-   Monoamine oxidase B-inhibitors: selegiline (e.g. Deprenyl®)    Anticholinergics-   Benzatropine (Cogentinol®), trihexyphenidyl (e.g. Artane®),    biperiden (e.g. Akineton®)    Glutamate/NMDA receptor antagonists-   Amantadine (e.g. PK-Merz®), memantine.

The precursor substance of the dopamine, the L-DOPA, is to be mentionedas first known dopaminergic. Contrary to dopamine, it can pass thehemato-encephalic barrier, then gets incorporated by the dopaminergicnerve cell and converted to dopamine by the cytosolic decarboxylase.L-DOPA is always combined with a decarboxylase inhibitor, that does notpenetrate into the brain, such as benserazide (in Madopar®) or carbidopa(e.g. Nacom®) to inhibit a peripheral conversion to dopamine. In thiscombination L-DOPA is still considered as the most effective and themost compatible dopamimetic substance.

The dopamine agonists, which directly influence the striatal dopaminereceptors, are another class of dopaminergics. The efficiency of allagonists is weaker than of L-DOPA, however, with these substances,complications such as cardiovascular complications as well as amentiaand hallucinations appear more frequently, besides the known sideeffects of L-DOPA. For this reason, these substances have to be appliedwith caution to senior, multimorbid patients.

Selegiline (e.g. Movergan®, Deprenyl®) inhibits irreversibly thedopamine degrading monoamine oxidase B and leads to an increase of thedopamine concentration in the striatum. Its effect is weak. In the firstyear of treatment a neuroprotective effect could be shown which washowever not detectable during the long-term treatment any more.

Anticholinergics reduce the resting tremor and the rigor, thehypokinesia is not influenced significantly. Due to their spectra ofside effects (amentia up to psychosis, glaucoma, anuresis, vertigo,fatigue) anticholinergics should be applied only in case of severeindication to patients above the age of 65 years, to patients withcognitive deficiencies not at all.

The amantadines (e.g. PK-Merz®) and the mere weakly efficient memantineare available as glutamate/NMDA receptor antagonists. The effects ofthese two substance groups are weak in comparison to the above describeddopaminergics.

Motoric late complications appear in many patients after long lastingapplication of dopaminergics as already mentioned above. A stabilisationof the blood level of L-DOPA (e.g. by application of retardpreparations) is reasonable for treatment of these side effects. Furtheron, the reduction of the L-DOPA dose rate and higher dosage of adopamine agonist preferably with long half-life (e.g. pergolide) isreasonable. A monotherapy can be attempted casually with an agonist(also in combination with amantadine). During stressing long-term offphases with painful dystonias the subcutaneous application of thedopamine agonist apomorphine cannot be abandoned in case of somepatients. Many times the treatment of these late complications isoverall unsatisfactory and requires an intensive cooperation of patientand physician. A stereotactic surgery can be considered as last option.

During dopaminergic medication the occurrence of psychoses is anadditional complication. The incidence and gravity of this complicationis determined by the age and possibly by other lesions of the brain,e.g. a vascular encephalopathy, in addition to the daily dose rate ofthe dopaminergic. Initially these medicament induced psychoses expressthemselves by unusually active dreams, illusionary cognitions andafterwards by visual, rarely also acoustical hallucinations anddelusions (Dr. med. A. Storch, Dr. med. J. Schwarz, “Geriatrie Praxis”,MMV Medizin Verlag GmbH München, Jahrgang 9 (1997) 4, 24–28).

Also surgical therapies of Parkinson's disease (transplantation ofdopamine synthesizing cells into the striatum; intrathecal applicationof neurotrophic factors; structural or functional lesion of overactivecore areas of the basal ganglions) are pursued in addition, whichhowever do not represent an alternative to the medicamentous treatment,because of partially serious side effects.

The object of the present invention is to provide physiologically wellcompatible substances, which can be applied for prophylaxis and/ortherapy of neurodegenerative diseases and furthermore avoid or at leastreduce the side effects of the known medicaments.

This object is solved by the technical teaching of the independent claim1 as well as the subject matters of the claims 5, 6, 9, and 11 of thepresent invention. Other favorable embodiments, aspects and details ofthe invention are evident from the dependent claims, the description andthe examples.

Surprisingly it was found, that(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid as well as(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and also the saltsof these compounds can be used according to the invention forprophylaxis and/or therapy of neurodegenerative diseases. Parkinson'sdisease, Huntington's chorea, cerebral circulatory disorders, ischemia,cerebral ischemia, apoplexy, cerebral apoplexy, brain deficiencies,senile dementia or Alzheimer's disease shall be mentioned as examples ofneurodegenerative diseases.

The salts of the above mentioned compound can be prepared by addition ofbasic solutions. Suitable bases are, for instance, alkaline and alkalineearth hydroxides, carbonates, hydrogen carbonates, phosphates or amines.

Salts of (2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid with amino acids areespecially suitable.

Alanine, asparagine, cysteine, glutamine, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, arginine, serine,ornithine, threonine, valine, tryptophan, tyrosine or derivatives ofthese amino acids can be used as amino acids. Basic amino acids such aslysine, arginine or histidine are preferred. Moreover basic salts ofamino acids such as sodium, potassium or lithium salts of amino acids ordisodium, dipotassium or dilithium salts of glutaminic acid orarparaginic acid are preferred.

In addition to amino acids, salts can be formed also with alkaline andalkaline earth cations or transition metal ions. Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺,Be²⁺, Mg²⁺, Ca²⁺, Sc³⁺, Mn²⁺, Fe³⁺, Cu⁺, Cu²⁺, Ag⁺ and Zn²⁺ can be used,preferred are Li⁺, Na⁺, K⁺, Cs⁺, Mg²⁺, Ca²⁺ and Zn²⁺ and especiallypreferred are Li⁺ and Na⁺.

Especially preferred are the lysine salts of(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid.

The term (2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid (2-MTDC)represents a diastereomeric mixture of the(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and the(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid.

From DE-A-21 16 629 the synthesis of not diastereomerically pure2-methylthiazolidine-2,4-dicarboxylic acid (2-MTDC), its use as ahepatoprotective agent as well as the preparation of notdiastereomerically 2-methylthiazolidine-2,4-dicarboxylic acid, which iscontained in pharmaceutical products in form of coated tablets orointments is known. Not diastereomerically pure 2-MTDC was proposed forsome applications as a pharmaceutical. The European patent applicationNo. 989 16 811 sets forth the use of not diastereomerically pure 2-MTDCas a mucolytic agent and the European patent application No. 989 16 809describes a combination preparation from not diastereomerically pure2-MTDC and paracetamol. The use of salts of 2-MTDC is preferred forprophylaxis and/or treatment of neurodegenerative diseases, especiallythe salts with amino acids, whereas the lysine salts are preferred.Preferred as well is the use of diastereomerically pure(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid or(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid in form of theirsalts, especially the salts with amino acids.

Thus, according to invention the salts of 2-MTDC,(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid can be used forpreparation of a pharmaceutic formulation for prophylaxis and/ortreatment of neurodegenerative diseases, especially of Parkinson'sdisease, Huntington's chorea, cerebral circulatory disorders, ischemia,cerebral ischemia, apoplexy, cerebral apoplexy, brain deficiencies,senile dementia or Alzheimer's disease. Besides the salts also the saltfree compounds (2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid as well as(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid are suitable forthis application.

Experiments were carried out for evidence of the activity of the freecompounds as well as of the salts of 2-MTDC,(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid, which shoulddetermine the effects of lysine salts onto the concentrations ofdopamine in the substantia nigra and the striatum, its metabolites3,4-dihyroxyphenylacetic acid (DOPAC) and homovanillinic acid (HVA) andonto 5-hydroxytryptamine (5-HT, serotonine) and its metabolite5-hydroxyindoleacetic acid (5-HIAA).

Furthermore, the concentration of 3-methoxytrytamine was measured, anindicator substance for the in vivo activity of dopaminergic neurons. Itis generated extraneuronally from dopamine via methylation of thehydroxyl group in position 3 by the catechol-O-methyltransferase (COMT).This means, that only dopamine, which was released during the activityof the dopaminergic nerve cells, can be the substrate of the enzyme. Themore active these are the higher is the concentration of3-methoxytyramine. The lysine salt of 2-MTDC was injectedintraperitoneally four to five days before the measuring of3-methoxytyramine.

It could be proven, that the lysine salt of 2-MTDC leads to an intenseactivation of the dopaminergic neurons. The inactivating effect ofmalonate is overcompensated by this effect. Thus, this observation isexciting, because the effect of the lysine salt of 2-MTDC is longpersistent.

To investigate dopaminergic neurons which sensitively react as is knownto neurotoxins what can lead e.g. to the Parkinson's disease the leftand the right striatum were selected as brain regions. The dopaminergiccell bodies are localized in the substantia nigra and project theiraxons into the dorsal striatum. The beneath described execution of theexperiments allows to investigate separately degenerative processes inthe region of the cell appendages which normally degenerate first and inthe cell bodies of the dopaminergic neurons which are more resistantcompared to noxa.

As neurotoxic substance sodium malonate was chosen, because thismitochondrial toxin leads to an acute release of dopamine in thestriatum of rats. Seven days after injecting the malonate it was proventhat the concentration of dopamine decreased to 6% in the striatumcompared to the contralateral striatum of the same animal. The effect ofmalonate on the dopamine concentration depends on time and dose rate.

Malonate is a competitive inhibitor of the succinate dehydrogenase(SDH). This mitochondrial enzyme plays a decisive role in the neuronalenergy supply. It is involved in the tricarbonic acid cycle and in theoxidative phosphorylation. An intrastriatal injection of malonateinitiates a lesion which appears excitotoxic and can be prevented viaapplication of competitive and non competitive antagonists at the NMDAreceptor. An intrastriatal injection of higher dose rates of malonateleads to lesions of dopaminergic and glutamatergic cell ends and to aconsiderable decrease of the striatal ATP concentrations and theconcentration of dopamine. The excitotoxic processes activated bymalonate which can lead to cell death are coupled to a bioenergeticdeficiency. Both processes are supposed to participate in thepathogenesis of numerous neurodegenerative diseases such as Alzheimer'sdisease, Huntington's chorea and Parkinson's disease.

According to the invention, the salt free compounds and the salts of2-MTDC, (2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid can be used directlyas salt free compound and salts respectively or manufactured aspharmaceutic formulation for prophylaxis and/or treatment ofneurodegenerative diseases especially of Parkinson's disease,Huntington's chorea, cerebral circulatory disorders, ischemia, cerebralischemia, apoplexy, cerebral apoplexy, brain deficiencies, seniledementia or Alzheimer's disease by administering a therapeuticallyeffective amount of the corresponding salt free compound and thecorresponding salt respectively to an individual suitable forprophylaxis and/or treatment of the particular neurodegenerativedisease.

The term ischemia refers to the decrease or the interruption of theblood circulation of an organ, organ part or tissue due to insufficientarterial blood supply (e.g. by thrombosis, embolism, endarteriitisobliterans, vascular spasm or tumors). Thus cerebral ischemia signifiesthe decrease or the interruption of the brain's blood circulation. Theconsequences of ischemia can be hypoxia, infarction as well as necrosis.

Another object of the present invention are pharmaceutic formulationscomprising the compounds (2R,4R)-2-methylthiazolidine-2,4-dicarboxylicacid, (2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or salts ofthese compounds as pharmaceutically active agent optionally togetherwith physiologically compatible carriers, auxiliary materials, fillers,flavor or color additives and/or solvents as well as diluents.

The compounds applicable according to invention as well as thepharmaceutic formulations applicable according to invention are suitablefor an intravenous, intraperitoneal, intramuscular, subcutaneous,rectal, transdermal, oral, nasal, buccal, sublingual or any otherapplication. Especially preferred is the oral and parenteraladministration of the compounds according to the invention.

The compounds according to the invention are applied in a dose rate from1 to 10,000 mg, preferred 10 to 5,000 mg and especially preferred from50 to 1,000 mg.

The pharmaceuticals according to the invention are prepared withconventional solid or liquid carriers or diluents and the conventionallyutilized pharmaceutical auxiliary materials corresponding to the desiredtype of application with a suitable dosage in a known manner. Suchpharmaceutical forms are e.g. tablets, film-coated tablets, coatedtablets, capsules, pills, powders, solutions, dispersions, suspensions,depot forms or inhalation solutions.

Also parenteral formulations such as injection or infusion solutionscome of course into consideration. Further on, formulations such assuppositories shall be mentioned.

Corresponding tablets can be obtained, for instance, by mixing thecompounds according to invention with known auxiliary materials forexample inert diluents such as dextrose, sugar, sorbitol, mannite,polyvinylpyrrolidone, blasting agents such as corn starch or alginicacid, adhesive agents such as starch or gelatine, lubricants such asmagnesium stearate or talc and/or agents for achieving a repositoryeffect such as carboxypolymethylene, carboxymethyl cellulose, celluloseacetatephthalate or polyvinylacetate. The tablets can be composed ofseveral layers.

Accordingly coated tablets can be prepared by coating of cores whichwere manufactured analogous to the tablets with agents typically used incoated tablet coatings such as polyvinylpyrrolidone or shellac, rubberarabicum, talc, titan dioxide or sugar. Thereby also the coated tabletcover can be composed of several layers whereas the auxiliary materialsmentioned in case of the tablets above can be utilized.

In addition, solutions or suspensions comprising the active agent whichcan be used according to the invention can contain flavor ameliorativeagents such as saccharine, cyclamate or sugar as well as flavoringsubstances such as vanillin or orange essence. Furthermore, they cancontain suspension auxiliary materials such as sodium carboxymethylcellulose or preservatives such as p-hydroxybenzoate. Capsules whichcontain active agents can be prepared, for example, by mixing the activeagent with an inert substrate such as lactose or sorbitol andencapsulating them into gelatine capsules.

Suitable suppositories can be prepared, for example, by mixing withsubstrates provided therefor such as neutral greases or polyethyleneglycol and derivatives thereof respectively.

Methods for preparing several formulations as well as the differentapplication techniques are known to the person skilled in the art anddescribed in detail, for instance, in “Remington's PharmaceuticalSciences, Mack publishing Co., Easton Pa.”.

EXAMPLES

The term 2-MTDC refers to (2R,4R)-2-methylthiazolidine-2,4-dicarboxylicacid, (2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid as well as(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid, whereas 2-MTDChowever preferably refers to the racemic compound.

Example 1

Preparation of the Lysine Salt of 2-MTDC

1. Prearrangement of a 4 N Potassium Acetate Solution:

294.45 g (3 mol) potassium acetate (pure, DAB) are resolved in approx.400 ml of demineralized water whereas the solution warms and is adjustedto an end volume of 750 ml after cooling down to 25° C. (±5° C.) in a 1l measuring cylinder.

2. Reaction:

In a 4 l round-bottomed flask 394.03 g (2.5 mol) cys*HCl in 545 ml MeOHare suspended under stirring and 1.2 l of isopropyl alcohol is added.First about 125 ml 4 N potassium acetate solution is added. Considerableamounts of a white precipitate are formed. To this suspension 243 ml(±2.5 ml) pyruvic acid are added. The utilized measuring cylinder isrinsed with 75 ml of isopropyl alcohol in total. Immediately afteraddition of the pyruvic acid the residual amount of 4 N potassiumacetate solution is added, the well stirable suspension warms up toapprox. 40° C. and during 4 h the temperature decreases slowly to RT.The stirring is stopped and the reaction mixture is kept at 4° C. for 16hours (i.e. over night). In doing so the precipitate deposits and alightly yellow supernatant is received.

3. Purification

The precipitate is filtered under suction via a Büchner funnel, pressedto dryness and is taken up two times in approx. 150 ml of demineralizedwater, filtered under suction and pressed to dryness. High vacuum ovendesiccation follows at approx. 40° C.

The raw product is added to approx. 900 ml of boiling demineralizedwater under intensive stirring (no magnetic stirrer!). It is heatedcarefully for approx. 10 minutes, whereas the viscous slurry is stirredintensively. Afterwards the suspension is cooled and filtered afterstanding for 3 hours under suction, pressed to dryness, taken up inapprox. 150 ml of ice cold demineralized water, filtered under suctionand again pressed to dryness. Afterwards high vacuum oven desiccationfollows at approx. 40° C. until constant mass (cold trap change). Theobtained product is crushed.

Yield: 354 g (74% of theory)

4. Lysine Salt

10 mmol of MTDC and 20 mmol of L-lysine base are dissolved in 20 ml ofwater at room temperature. The transparent solution is then lyophilized.In the solution no pyruvic acid or free cysteine could be detected.

Example 2

Animal Experiments:

In a test series 2×0.8 mmol/kg 2-MTDC lysine salt (2-MTDC-lys) wasapplied intraperitoneally to rats after 12 h and 48 h. 30 minutes afterthe last application the one half of the rats received 2 μmol of sodiummalonate into the left striatum via an afore stereotactically implantedguide cannula. The right striatum was used as intraindividual controltissue. Four days after the last application the concentrations of thetransmitters and of their metabolites were measured.

Another group of rats received solvents into the left striatum and athird group received 2 μmol of sodium malonate into the left striatum.

Description of the Experiments:

A group of 6 Wistar rats, Charles River, Sulzbach Rosenberg receive twotimes intraperitoneally applied 0.8 mmol/5 ml/kg body weight 2-MTDC-lyswithin 48 h. 30 minutes after the second application 2 μmol of sodiummalonate (Sigma) dissolved in physiological common salt solution areapplied stereotactically during general anesthesia (ketamine 80 mg/kgand xylazine 6–10 mg/kg (IM)) via a syringe with an attached precisionpump directly into the left striatum (flow rate: 0.5 μl/min, totalvolume 2 μl).

Four days after the last application the striatum and the substantianigra of the treated side as well as of the other side is excisedseparately in each case, balanced and the tissue is homogenized in 0.1molar perchloric acid (a striatum in 500 μl, a substantia nigra in 150μl) and is centrifuged afterwards at 13,000 rpm for 5 minutes (Biofuge13, Heraeus). The supernatant is filtrated at 10,000 rpm for 10 minutes(Millipore, 0.22 μm pore size). Aliquots of the eluent are injecteddirectly into the HPLC apparatus. Dopamine, DOPAC, HVA, 5-HT, 5HIAA and3-methoxytyramine are separated via HPLC-ELCD and quantifiedcolorimetrically. As the concentration of dopamine relative to theconcentration of the metabolites is high in the striatum dopamine ismeasured in the striatum separately (conditions: 2 μl injection volume,detector adjusted to 20 nAmp).

The conditions for the other substances and the substantia nigra are asfollows: 5 μl injection volume, detector: 5 nAmp.

Another group of rats receives intraperitoneally solvents within 48 h(four times 50% propanediol as well as two times physiological commonsalt solution in each case 5 ml/kg) and 30 minutes after the lastapplication 2 μmol sodium malonate is injected intrastriatally left. Thesecond test procedure is equivalent to the above described.

A third group of rats receives intraperitoneally solvents within 48 h(four times 50% propanediol and two times physiological common saltsolution in each case 5 ml/kg) and 30 minutes after the last application2 μl physiological common salt solution is injected into the leftstriatum. The further test procedure is equivalent to the abovedescribed.

Results:

The lysine salt of 2-MTDC (2-MTDC-lys) solely has no influence on theconcentration of dopamine, DOPAC, HVA. A pretreatment with 2-MTDC-lyshowever prevents the toxic effect of malonate. 2-MTDC-lys actsneuroprotectively as it prevents the effect of malonate on the reductionof the dopamine and dopamine metabolites concentrations respectively.

In the chosen dose rate and during the examination period of four dayscalculated from the last application 2-MTDC-lys affects obviouslydopaminergic neurons but not serotonergic neurons. The effects ontodopaminergic neurons comprise a long persistent activation which isprobably also the reason for the neuroprotection. The treatment with2-MTDC-lys prevents the neurodegeneration as well of the nerve ends inthe striatum as of the cell bodies in the substantia nigra(prophylactically neuroprotective).

The test series of example 2 was repeated whereas the lysine salt of2-MTDC was replaced by the sodium salt. It could be shown that also thesodium salt of 2-MTDC provides comparable results.

Example 3 Therapeutic Effect of 2-MTDC and Salts Thereof

In another animal experiment model (apoplexy model) it could be shown(examples 3 and 4) that 2-MTDC as well as salts of 2-MTDC have acerebroprotective effect. As well the free acids of 2-MTDC[(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid] as, for instance,the lysine salts of 2-MTDC are capable of reducing the infarction extentinduced by occlusion of the cerebral artery in mice (MNRI mice). Theseresults are described in the following on the example of the sodium saltof 2-MTDC.

Sodium Salts of 2-MTDC (2-MTDC-Na)

The following test series deals with the examination of theneuroprotective effect of 2-MTDC-Na in application of 2-MTDC-Na afterthe ischemia.

Description of the Experiments

Per test group 13 through 16 MNRI mice were employed. The middlecerebral artery of the mice was occluded according to the method ofWelsh (F. A. Welsh, T. Sakamoto, A. E. McKee, R. E. Sims, J. Neurochem.1987, 49, 846–851). For that purpose the mice are anesthetized withtribromoethanol (600 mg/kg body weight, intraperitoneally). Immediatelyafter the anesthesia a small cavity was drilled into the mice cranium tolay open the middle cerebral artery. The strain as well as both arms ofthe middle cerebral artery were occluded via electro coagulation.Meanwhile the blood heat of the mice was kept on 37° C.±1° C. byutilizing a heating lamp. Subsequently the mice were kept at asurrounding temperature of 30° C. for another 2 h.

One group of mice was injected intraperitoneally 2-MTDC-Na (200 mg/kg2-MTDC-Na dissolved in 0.9% NaCl solution) 15 minutes after and anothergroup 60 minutes after the occlusion of the middle cerebral artery. Athird group received the vehicle only (0.9% NaCl solution without2-MTDC-Na).

For the following histologic examination 2 days after the occlusion ofthe middle cerebral artery the mice were anesthetized again withtribromoethanol and received intraperitoneally applied 0.5 ml of a 1.5%solution of neutral red (toluylene red, Acros Chimica) in addition. Thebrains were extracted and kept for 24 h in a fixative solution (4%formalin with phosphate buffer at pH 7.4).

The tissue on the surface of the brain which was not colored by neutralred was measured as surface region affected by infarction underutilizing a color analysis system (Kontron, Eching, Germany) (C.Backhauβ, C. Karkoutly, M. Welsh, J. Krieglstein, J. Pharmacol ToxicolMethods, 1992, 27 (2), 27–32).

Results

Infarction formation could only be proved at the cortical tissue.Moreover, the infarction volume correlated with the infarction surface.2-MTDC-Na was capable of reducing significantly the area of the brainwhich was affected by the infarction whereas it seems to be important toapply 2-MTDC-Na after the occlusion of the middle cerebral artery assoon as possible. In the test series where 2-MTDC-Na was injected onehour after the occlusion of the middle cerebral artery only a stronglyattenuated effect of the infarction reduction was shown.

Thus, it could be shown that 2-MTDC-Na administered after the ischemiaexhibits further on its neuroprotective effect whereas the therapeuticalwindow for the application of 2-MTDC-Na is about 60 minutes after theocclusion of the middle cerebral artery.

Example 4 Protective Effect of 2-MTDC and Salts Thereof

The execution of the test according to example 3 was repeated whereas2-MTDC-Na was applied one hour before the occlusion of the middlecerebral artery (MCA-O: occlusion of the middle cerebral artery).

It could be shown that 2-MTDC-Na protects the brain of the animalsagainst damages which were caused by cerebral ischemia, if 1 h beforethe occlusion of the middle cerebral artery the 2-MTDC-Na is injectedintraperitoneally at a concentration of 200 mg/kg body weight of thetest animal.

The test series according to example 3 and 4 were repeated with2-MTDC-lys as well as with the free acid of 2-MTDC instead of 2-MTDC-Na.The experiments with 2-MTDC-lys and the free acid of 2-MTDC producedsimilar results as the experiments with 2-MTDC-Na.

1. A method of treating neurodegenerative diseases selected from thegroup consisting of Parkinson, Huntington's chorea, cerebral circulatorydisorders, ischemia, cerebral ischemia, apoplexy, cerebral apoplexy,senile dementia, and Alzheimer's disease, comprising administering(2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid and/or(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid as well as theirsalts to a patient in need thereof.
 2. The method, according to claim 1wherein (2R,4R)-2-methylthiazolidine-2,4-dicarboxylic acid,(2S,4R)-2-methylthiazolidine-2,4-dicarboxylic acid, and/or(2RS,4R)-2-methylthiazolidine-2,4-dicarboxylic acid is administered in adose rate of 1–10,000 mg, especially from 10–5,000mg.